Cryptococcal meningitis is one of the most important opportunistic infections affecting people living with HIV/AIDS. Recent epidemiologic studies indicate that approximately 1 million new cases occur each year and over 700,000 people die each year; in sub-Saharan Africa, more HIV patients die of cryptococcal meningitis each year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B combined with 5-flucytosine. Although this regimen is easily administered in resource-rich regions of the world, it is not available in many resource-limited regions with the highest burden of disease. In these regions, fluconazole, a less effective, fungistatic agent, is used because it is readily available, safe, and administered orally. Unfortunately, patients treated with FLU have much higher death rates; this has been attributed to the fungistatic activity of FLU. Thus, new therapies for cryptococcosis are urgently needed and this is the long-term goal of our project. Because molecules with fungicidal activity are clinically superior to fungistatic drugs, we have developed a high throughput screening assay that directly identifies molecules with fungicidal activity toward C. neoformans. We have screened two distinct libraries with the assay: 1) a 350,000 compound library through an NIH-funded Molecular Probes and Library Screening Network project and 2) a focused set of 700 molecules from Merck Laboratories collection of anti-candidal molecules. The former screen was designed to identify a molecule with activity against cell wall related processes in C. neoformans: a novel benzimidazole thiourea scaffold with low human toxicity and good anti-cryptococcal activity emerged from this screen. We will identify its mechanism of action in Aim 1 to set the stage for subsequent optimization of its anti-cryptococcal activity. From the Merck Library, we have identified 22 molecules with fungicidal activity against C. neoformans at concentrations ? 2 g/mL. We will complete the secondary screening of the set of Merck library hits in Aim 2 and identify the mechanism of action for a tota of four high priority scaffolds. Initial evaluation indicates that a set of squalene synthase inhibitors are among the hits. We will test that hypothesis and identify the target of three high priority scaffolds from the remaining hits. The mechanistic information that emerges from this exploratory project will be used as a basis for a subsequent application to optimize the anti-cryptococcal activity of the scaffolds.